NCLX expression regulation by mild stress conditions

Abstract

Mitochondrial matrix Ca2+ efflux is largely mediated by the mitochondrial Na+/Ca2+ exchanger (NCLX), directly coupling mitochondrial Ca2+ and Na+ handling. NCLX has been shown to play a central role in many pathophysiological processes, including colorectal tumor metastasis, and Parkinson’s and Alzheimer diseases. Indeed, sporadic Alzheimer´s disease patients had decreased frontal cortex NCLX levels, an effect recapitulated in mouse models. However, transcriptional mechanisms specifying its functional states has not been clearly defined. Here, we aimed to investigate how NCLX expression is regulated. NCLX mRNA was consistently found to be increased after mild stressful events, including scrambled siRNA transfection and control adenoviral vector transduction. We then hypothesized that NCLX gene expression was controlled by a stress‐sensitive pathway. Indeed, serum‐deprived astrocytes and mouse embryonic fibroblasts (MEFs) showed an increase in NCLX mRNA levels, an effect that was even further modulated by incubation with thapsigargin or H2O2, indicating expression sensitivity to a wide variety of stress signals. Here, we took advantage of a genome‐wide approach using available epigenomics datasets, and transcriptomic data to pinpoint transcriptional factors that orchestrate NCLX gene expression upon homeostatic conditions in astrocytes. We identified promoter‐distal enhancer‐like elements in forebrain mouse and primary astrocytes using chromatin immunoprecipitation high‐throughput sequencing (ChIP‐seq) data marked by histone acetylation of lysine 27 (H3K27ac). We also used a public ATAC‐seq to define a coincident region with accessible chromatin. Still, transcription factor binding sites uncovered through sequence‐based single site analysis of the NCLX gene indicated several putative transcription factors, including many components of the NF‐κB family. In parallel, public RNA‐seq databases were mined for patterns that were correlated with transcription factor known effects, before proceeding with binding confirmation through ChIP‐PCR. In summary, we were able to build a foundation to test the modulation of NCLX expression. This proof‐of‐concept of NCLX stress‐induced mechanism may be further explored as a tool for targeting cellular Na+/Ca2+signaling, mitochondrial function, and metabolism.